This invention relates generally to the separation of components of a fermentation broth and, more specifically to the isolation of water miscible compounds having boiling points higher than water from other fermentation broth components.
Environmental and cost reduction incentives exist to design process schemes that have the ability to separate and optionally recycle components in a fermentation including the cell mass, residual media and media salts, residual substrate such as sucrose and/or glucose, and water. Efforts have also been made to recycle cell mass as a means to improve the fermentation productivity. Less effort has been made in the area of recovering the residual media and media salts for reuse in the fermentation. In this regard, most efforts have focused on reducing initial media costs, rather than downstream recovery. The resulting “low cost” media is often not optimal for cell growth and product production. By developing effective methods for the recovery of media components, a more optimal media recipe can be utilized with fewer restrictions on initial raw material costs.
The isolation of compounds on large scale with useful purity is a complex challenge in process chemistry. Differences in scale alone can render isolation procedures developed on laboratory benchtop scale impractical or even not viable at pilot or commercial scales. Isolation of compounds from complex mixtures depends on numerous factors including whether the compound is a solid or liquid at ambient temperatures, the compounds boiling point, density, polarity, the presence or absence of pH sensitive functional groups, and solubility in organic solvents versus water. These factors also apply to all other components of the mixture from which the compound of interest is to be isolated. Another property that factors into isolation of a compound, organic compounds in particular, is how it partitions between two immiscible phases, such as between water and an organic solvent. Compounds that are particularly polar are often more soluble in water than in common organic solvents used in extraction processes. Some compounds are particularly challenging to isolate from water by extractive methods due to their amphiphilic character. Amphiphiles are compounds that possess both a polar portion and a lipophilic portion. These compounds can complicate isolation by extraction by causing intractable emulsions.
Moreover, when a compound is prepared from a fermentation the amount of water can be substantially higher than the compound of interest, requiring isolation of a minor component from a complex mixture. Isolation of compounds that boil at a higher temperature than water further adds to the complexity and cost of the separation since the compound cannot be distilled directly from the fermentation broth as is the case, for example, in an ethanol fermentation process. In this regard, interactions between the compound of interest and water can cause the two entities to co-distill as an azeotrope at a boiling point different from the two purified components. Azeotrope formation is not readily predictable. This can diminish recovery of the compound of interest when trying to separate it from water. When a compound has polar functional groups another concern is how it may interact with other compounds present in the water phase, including any salts and metal ions, for example.
The nature of the functional groups present in a compound of interest can complicate the separation of salts. For example, one or more functional groups of a compound can interact with or chelate cations or anions. Chelation occurs in a size dependent manner with respect to the cation or anion and is also dependent on the disposition of the functional groups on the compound of interest. Chelation and other interactions can render some salts soluble in a liquid compound even in the absence of water, while other salts can be insoluble in the absence of water despite the presence of a compound with functional groups capable of interacting with salts. These types of effects on salt solubility are difficult to predict. Further adding to the complexity of the interaction between a compound and salts, is the nature of any co-solvents. For example, during the isolation of a compound of interest that is water miscible, hydrogen bonding and other interactions with water can disrupt the interaction between the salts and the compound of interest. Thus, in some cases a salt can be separated more readily from a compound in the presence of some amount of water. However, the amount of water that balances salt supersaturation allowing salt separation by crystallization, for example, while maintaining water's ability to disrupt chelation and other interactions between a compound of interest and any salts is difficult to predict.
Yet a further challenge in developing isolation methods is the potential reactivity of biosynthetic byproducts such as organic acids, excess substrate, and the like. Under conditions of heating, excess substrate can degrade and cause undesirable coloration of product. Additionally, some byproducts can react with the product of interest, effectively lowering isolation yields. These byproducts can include those formed during fermentation as well as byproducts formed during steps of the isolation procedure itself, for example due to degradation processes at elevated temperatures during a distillation, water evaporation, and the like.
Thus, there is a need to develop processes that allow for the isolation of water miscible compounds that have boiling points higher than water from microbial fermentations, while bearing in mind the environmental and cost benefit of recycling other fermentation components. The present invention satisfies this need and provides related advantages as well.